Reaction Mechanism of Fructose-2,6-bisphosphatase

Mizuguchi, Hiroyuki; Cook, Paul F.; Tai, Cheng‐Chi; Hasemann, Charles A.; Uyeda, Kosaku

doi:10.1074/jbc.274.4.2166

Cited by 38 publications

(29 citation statements)

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“…This model is consistent with previous kinetic experiments where it has been reported that the rate of phosphoenzyme formation is greater than the overall reaction rate (33), that Fru-6-P inhibits the Fru-2,6-Pase reaction (34), that Fru-6-P release is the rate-limiting step in the overall reaction (35), and that P i accelerates E-P hydrolysis by competing for Fru-6-P binding (presumably at the 6-phosphate binding site) (33). In our own studies, we showed that Fru-6-P release and phosphohistidine breakdown occur in parallel in the wild type enzyme (1). Together, the structural and kinetic data indicate that E-P hydrolysis does not occur until after Fru-6-P has dissociated from the enzyme, and that E-P hydrolysis is fast (Scheme 1).…”

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confidence: 73%

“…This enzyme unexpectedly retains 17% of the RT2K-Wo Fru-2,6-Pase activity and led to a series of experiments to assess the basis for this activity. The results of mutagenesis and kinetic analyses on the Fru-2,6-Pase domain of the RT2K isozyme are presented in a companion paper (1). We report here the crystallographic structure of the Fru-2,6-Pase domain of the RT2K H256A mutant enzyme.…”

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confidence: 99%

“…of Internal Medicine, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-8884. 1 The abbreviations used are: Fru-6-P,2-kinase, fructose-6-phosphate,2-kinase; Fru-2,6-Pase, fructose-2,6-bisphosphatase; Fru-2,6-P 2 , fructose-2,6-bisphosphate; Fru-6-P, ␤-D-fructose-6-phosphate; AMP-PNP, 5Ј-adenylyl imidodiphosphate; RL2K, rat liver isozyme of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase; RT2K, rat testis isozyme of fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase; RT2K-Wo, RT2K with all four tryptophans mutated to phenylala-His 390 , which is neither coupled to Glu 325 , nor in a position to donate a proton to Fru-6-P. It is important to note, however, that neither the RT2K-Wo nor the original RL2K Fru-2,6-Pase domain structures contained bound ligands (Fru-6-P or Fru-2,6-P 2 ), which has hampered a thorough characterization of the Fru-2,6-Pase mechanism.…”

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confidence: 99%

“…There have been several models of Fru-2,6-Pase catalysis proposed, based first on mutagenesis directed by the homology of the Fru-2,6-Pase domain to the phosphoglycerate mutase family (16,17) and later based on crystal structures (1,7,8,21 (1,13,14,21). His 256 is perfectly placed for an in-line attack that would break the O-2-P-2 bond of Fru-2,6-P 2 with an inversion of phosphate geometry (Fig.…”

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Crystal Structure of the H256A Mutant of Rat Testis Fructose-6-phosphate,2-kinase/Fructose-2,6-bisphosphatase

Yuen¹,

Mizuguchi

Lee³

et al. 1999

Journal of Biological Chemistry

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Fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase (Fru-6-P,2-kinase/Fru-2,6-Pase) is a bifunctional enzyme, catalyzing the interconversion of ␤-D-fructose-6-phosphate (Fru-6-P) and fructose-2,6-bisphosphate (Fru-2,6-P 2 ) at distinct active sites. A mutant rat testis isozyme with an alanine replacement for the catalytic histidine (H256A) in the Fru-2,6-Pase domain retains 17% of the wild type activity (Mizuguchi, H., Cook, P. F., Tai, C-H., Hasemann, C. A., and Uyeda, K. (1998) J. Biol. Chem. 274, 2166 -2175). We have solved the crystal structure of H256A to a resolution of 2.4 Å by molecular replacement. Clear electron density for Fru-6-P is found at the Fru-2,6-Pase active site, revealing the important interactions in substrate/product binding. A superposition of the H256A structure with the RT2K-Wo structure reveals no significant reorganization of the active site resulting from the binding of Fru-6-P or the H256A mutation. Using this superposition, we have built a view of the Fru-2,6-P 2 -bound enzyme and identify the residues responsible for catalysis. This analysis yields distinct catalytic mechanisms for the wild type and mutant proteins. The wild type mechanism would lead to an inefficient transfer of a proton to the leaving group Fru-6-P, which is consistent with a view of this event being rate-limiting, explaining the extremely slow turnover (0.032 s ؊1 ) of the Fru-2,6-Pase in all Fru-6-P,2-kinase/Fru-2,6-Pase isozymes.

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Crystal Structure of the H256A Mutant of Rat Testis Fructose-6-phosphate,2-kinase/Fructose-2,6-bisphosphatase

Yuen¹,

Mizuguchi

Lee³

et al. 1999

Journal of Biological Chemistry

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“…However, a more recent study found that the H258A mutant form of the rat liver FBPase-2 is an active bisphosphatase that catalyzes hydrolysis without formation of a covalent intermediate (36). In addition, residual catalytic activity for the H256A mutant has been reported for rat testis FBPase-2, which retains about 17% catalytic activity (37). Because of the formation of a phospho-histidine intermediate in the wild type enzymes, the hydrolysis reaction in the mutant enzymes has to occur by a different mechanism.…”

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confidence: 99%

Structural and Biochemical Studies of TIGAR (TP53-induced Glycolysis and Apoptosis Regulator)

Jogl

2009

Journal of Biological Chemistry

106

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Activation of the p53 tumor suppressor by cellular stress leads to variable responses ranging from growth inhibition to apoptosis. TIGAR is a novel p53-inducible gene that inhibits glycolysis by reducing cellular levels of fructose-2,6-bisphosphate, an activator of glycolysis and inhibitor of gluconeogenesis. Here we describe structural and biochemical studies of TIGAR from Danio rerio. The overall structure forms a histidine phosphatase fold with a phosphate molecule coordinated to the catalytic histidine residue and a second phosphate molecule in a position not observed in other phosphatases. The recombinant human and zebra fish enzymes hydrolyze fructose-2,6-bisphosphate as well as fructose-1,6-bisphosphate but not fructose 6-phosphate in vitro. The TIGAR active site is open and positively charged, consistent with its enzymatic function as bisphosphatase. The closest related structures are the bacterial broad specificity phosphatase PhoE and the fructose-2,6-bisphosphatase domain of the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. The structural comparison shows that TIGAR combines an accessible active site as observed in PhoE with a charged substrate-binding pocket as seen in the fructose-2,6-bisphosphatase domain of the bifunctional enzyme.The p53 tumor suppressor protein is a stress-induced transcription factor that controls various cellular response mechanisms including cell cycle arrest and apoptosis (1, 2). As part of a complex signaling network that includes positive and negative feedback loops, the p53 pathway determines cell death or survival after its activation by various forms of cellular damage (3). TIGAR (TP53-induced glycolysis and apoptosis regulator) is a recently identified p53-regulated gene that was found to lower fructose-2,6-bisphosphate (Fru-2,6-P 2 ) 2 levels in cells. TIGAR expression resulted in down-regulation of glycolysis, reduction of intracellular levels of reactive oxygen species, and protection from apoptosis (4).Fru-2,6-P 2 is a key regulator of cellular metabolism that activates glycolysis and inhibits gluconeogenesis. Fru-2,6-P 2 is found in almost all eukaryotes but is absent from prokaryotes (5). Other functions for this molecule in addition to its allosteric regulation of glycolysis and gluconeogenesis have been identified (6). The structure and function of the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (FBPase-2) enzymes responsible for Fru-2,6-P 2 synthesis and removal within the cell have been studied for more than 20 years (7). The transcription of a hypoxia-inducible isoform (PFKFB3) is controlled by hypoxia-inducible factor 1 (8), and this isoform is frequently found in cancer cells (9). Thus, synthesis and removal of Fru-2,6-P 2 by PFKFB3 and TIGAR is controlled by hypoxia-inducible factor 1 and p53, emphasizing the importance of metabolic regulation for tumor suppression (10).Histidine phosphatases share a strictly conserved catalytic core centered at a histidine residue that is phosphorylated during the reaction. Two ...

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Fructose 6‐Phosphate 2‐Kinase:Fructose 2,6‐Bisphosphatase

Uyeda

2002

Wiley Encyclopedia of Molecular Medicine

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Reaction Mechanism of Fructose-2,6-bisphosphatase

Cited by 38 publications

References 26 publications

Crystal Structure of the H256A Mutant of Rat Testis Fructose-6-phosphate,2-kinase/Fructose-2,6-bisphosphatase

Crystal Structure of the H256A Mutant of Rat Testis Fructose-6-phosphate,2-kinase/Fructose-2,6-bisphosphatase

Structural and Biochemical Studies of TIGAR (TP53-induced Glycolysis and Apoptosis Regulator)

Fructose 6‐Phosphate 2‐Kinase:Fructose 2,6‐Bisphosphatase

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